Hypothetical protein Rv2635 of Mycobacterium tuberculosis is an intrinsically disordered nucleoid-associated protein that undergoes phase separation.

Functional characterization of small hypothetical proteins of bacterial pathogens is challenging often due to species-specificity and the lack of homologous domains. Nearly one-third of the proteome of Mycobacterium tuberculosis, is represented by hypothetical proteins, of which ∼11 % are small proteins. In this study, we report the biochemical and functional features of Rv2635, a small hypothetical protein. Using in silico approaches and circular dichroism, we establish that Rv2635 is an intrinsically disordered protein (IDP). Significantly, GFP-tagged Rv2635 could undergo phase separation at a low salt concentration. FRAP analysis showed that the Rv2635 condensate formation was completely reversible. To get insights into the biological function, we explored the predicted DNA-binding ability of the protein. EMSA showed that the protein binds to DNA in a sequence-independent fashion. Overexpression of Rv2635 in E. coli caused bacterial nucleoid compaction and co-localization of Rv2635 with the condensed nucleoid. These results suggest that Rv2635 is a nucleoid-associated protein (NAP). Employing truncated versions of Rv2635, we demonstrate that its unstructured regions are indispensable for phase separation, whereas the C-terminal contributes in DNA-binding. Altogether, the findings demonstrate that Rv2635 is a phase-separating IDP that may function as a NAP in mycobacteria.